Microbiological and Clinical Outcomes of Methicillin-Susceptible Staphylococcus aureus Isolated in Lung Transplant Perioperative Donor and Recipient Respiratory Cultures

Staphylococcus aureus is one of the most common organisms isolated from respiratory secretions in lung transplant donors and recipients perioperatively. Within the first 90 days after lung transplantation, methicillin-susceptible Staphylococcus aureus (MSSA) infections have been associated with increased mortality and acute and chronic rejection. However, it is unclear whether respiratory cultures positive for MSSA at the time of transplantation can lead to clinically significant infection. The aim of this study was to assess the microbiological and clinical outcomes for lung transplant recipients (LTRs) with positive perioperative donor or/and recipient respiratory cultures for MSSA. A retrospective study was conducted evaluating MSSA-positive respiratory cultures at the time of lung transplantation from donors and/or recipients from January 1, 2008, to December 30, 2019. Patients who did not have a bronchoalveolar lavage at 2 weeks after the lung transplant or died within 2 weeks of lung transplant were excluded. The main outcome was MSSA eradication at 2-week bronchoscopy. Recipients were evaluated for MSSA infections at the 12-week period after the transplant. Of the 1,678 individuals who underwent lung transplantation, 218 LTRs had S. aureus isolated in perioperative donor or recipient respiratory cultures, and 29 were subsequently excluded. Of the remaining 189 LTRs, MSSA eradication at the 2-week bronchoscopy was achieved in 186 (98.4%) recipients. During the 12-week follow-up, 15 (7.9%) recipients were diagnosed with MSSA pneumonia; concurrent MSSA bacteremia was noted in one recipient. No anastomotic infection, empyema, or lung abscess related to MSSA was diagnosed during the follow-up period. In LTRs, the rate of MSSA eradication at 2-week post-transplant recipients is high, and it is associated with a low rate of infectious complication within the first 12 weeks after transplant. Most of the recipients received a combination therapy with at least one agent active against MSSA. More studies to evaluate the optimal antimicrobial stewardship policies regarding the regimen and duration of antibiotic therapy for these patients are needed.


Introduction
Infection in lung transplant recipients (LTRs) is a leading cause of morbidity and mortality 1,2,3 . Impaired graft mucociliary function, weak cough reflex, airway ischemia, periopera tive invasive procedures, and the level of immuno suppression put LTRs at increased risk of infection, particu larly pneumonia 4 . During the early posttransplant period, pneumonia develops mainly due to nosocomial acquisition, recipient colonization, or donorderived infection 5,6 . One of the most common organisms that has been implicated in positive respiratory cultures at the time of transplant is Staphylococcus aureus. Methicillinsusceptible Staphylo coccus aureus (MSSA) contributes up to 25% of positive respiratory cultures in LTRs and 48% in donors 7,8 .
Methicillinresistant S. aureus (MRSA) colonization at the time of lung transplant has been found to be a risk factor for progression to infection in early postlungtransplant period, but there is no established correlation between MSSA isolated in peritransplant respiratory culture and progression to infection 9 . At our center, piperacillintazobactam is used empirically for prophylaxis after lung transplantation for 72 hours until intraoperative culture results are available. The practice of most transplant physicians is to switch antibiotics to cefazolin or cloxacillin if MSSA is isolated in respiratory cultures and treat for at least 14 days with intravenous antibi otic therapy. In the case of multiple organisms being recov ered, or the recipient is known to be colonized with Pseudomonas, cefazolin or cloxacillin is merely added to piperacillintazobactam. This practice is mainly derived from studies of patients with MSSA bacteremia, where using an antistaphyloccocal penicillin or cefazolin is associated with significantly lower 30day mortality compared to piperacillintazobactam 10 .
The aim of the study was to assess the rate of eradication of MSSA isolated from recipient or donor respiratory cul tures at the time of lung transplantation and to assess the development of MSSArelated infection and complications during the first 12 weeks after the transplant.

Study Design and Patient Population
We conducted a retrospective observational cohort study at the Toronto General Hospital, a 456bed hospital in Toronto, Ontario, Canada. LTRs with MSSA cultured from bronchoal veolar lavage (BAL) from recipients, donors, or both at the time of lung transplantation were recruited from January 1, 2008, to December 30, 2019. During the study period, the routine practice at our center consisted of performing surveil lance bronchoscopy for all patients at 2 weeks, 6 weeks, and 12 weeks. All transplanted patients during the study time frame were eligible and were included if they met all the fol lowing criteria: (1) aged 18 years or older, (2) had followup surveillance bronchoscopy at 2 weeks, (3) survived for 2 weeks after lung transplant, and (4) if MSSA was isolated in the recipient, donor, or both. As mentioned, we categorized the source of the positive MSSA BAL culture into three cat egories: recipient, donor, and both donor and recipient. Patients were excluded if MRSA was cultured at the time of lung transplantation, in either the donor or recipient. The results of BAL cultures at 2 weeks, 6 weeks, and 12 weeks were assessed for MSSA eradication, persistence, recurrence, or reinfection, as defined in the following sections. Antibiotics used as a prophylaxis or treatment were assessed from the transplantation date through discharge date or 2 weeks after transplantation, whichever was longer.

Primary and Secondary Endpoints
The primary endpoint was MSSA eradication from BAL cul ture at the 2week bronchoscopy. The secondary endpoints included (1) MSSA eradication at 6 weeks and 12 weeks; (2) clinical outcome at the 2week followup as assessed by clin ical improvement, stability, or deterioration; and (3) clinical criteria of MSSA infection at followup that occurred by 12 weeks after lung transplantation (see Definitions) Definitions MSSA eradication was defined as a negative BAL culture for MSSA in the followup bronchoscopy at 2 weeks. MSSA per sistence was defined as a continuously positive BAL culture since the time of transplantation. MSSA recurrence was defined as a positive BAL culture after eradication for MSSA with the same antibiotic susceptibility testing (assumed to be the same strain) compared with the initial MSSA strain isolated at the time of transplantation. Reinfection was defined as a positive BAL culture for MSSA after eradication with a different antibi otic susceptibility (assumed to be a different strain) in compari son to the MSSA strain isolated at the time of transplantation.
The clinical assessment at 2 weeks was determined by improvement defined by a decrease in oxygen requirement, no ongoing signs of sepsis, and no new infiltration in the Chest Xray (CXR); stability was defined by stable oxygen supplement since starting the targeted therapy, no ongoing signs of sepsis, and no new infiltration in the CXR; and dete rioration by development of respiratory failure, fever or sep sis, or new infiltration in the CXR. MSSA infection within the first 12 weeks after lung transplant was defined according to the International Society for Heart and Lung Transplantation consensus criteria 11 .

Data Collection
Data were abstracted from the database of the Toronto Lung Transplant Program of Toronto General Hospital for all patients' cultures obtained at the time of transplantation and at 2 weeks, 6 weeks, and 12 weeks after transplantation. Paper and electronic patient charts were also examined for clinical data, laboratory data, vital signs, and diagnostic imaging. Collected data included patient demographics, underlying lung disease for lung transplant, comorbidities, antibiotic treatment, and clinical outcome.

Statistical Analysis
Characteristics were compared using Student's t test for con tinuous variables and chisquare or Fisher's exact tests for categorical variables. All analyses were performed using GraphPad Prism version 9.4.1 (GraphPad Software, San Diego, CA, USA)

Ethical Approval
The study protocol was approved by the Research Ethics Board of the University Health Network, Toronto, Ontario, Canada (REB approval number: 176107). Patient consent was waived for this retrospective cohort study.

Patient Demographics and Clinical Characteristics
During the study period of January 1, 2008, to December 31, 2019, 1,537 patients underwent lung transplantation, of whom 218 (14.2%) had positive respiratory cultures for S. aureus from BAL fluid at the time of transplantation. Twenty nine recipients were excluded due to death within 14 days of transplant (5), lack of followup bronchoscopy at 2 weeks (5), or isolation of MRSA (7 in the recipient and 5 in the donor) ( Figure 1). The baseline demographic and clinical characteristics for the cohort of all LTRs as well as the MSSA recipients are presented in Table 1. Compared to the recipi ents without MSSA, the MSSA group was significantly younger and more frequently male. No differences were observed in the type of transplantation or the underlying dis eases. For the MSSA group, a doublelung transplant was

Treatment Characteristics
All LTRs in our study had received empirical prophylactic antibiotics after transplantation. Piperacillintazobactam was the most frequently administered empirical therapy (70.3%), followed by meropenem (18.5%), and then ceftazidime (8.5%). One hundred and eighteen (62.4%) recipients received at least one dose of vancomycin empirically, in addition to a betalactam antibiotic, prior to the culture results being finalized.
With regard to targeted antibiotic therapy for MSSA, 98 (51.9%) received a combination of antibiotics, whereas 91 (48.1%) received monotherapy. The mean duration of antibiotic therapy for MSSA was 12.5 days. The empirical and targeted antibiotic regimens for the 189 recipients are shown in Table 2.

Microbiological Outcomes
The microbiological outcomes of the 189 patients are shown in Table 3. A total of 15 patients were documented with MSSA throughout the 12week observation period. The pri mary outcome of MSSA eradication at the 2week bronchos copy occurred in 186 (98.4%) recipients. However, at the

Clinical Outcome
At the 2week assessment, 150 (79.4%) recipients exhibited clinical improvement, while 29 (15.3%) recipients deterio rated, and 9 (4.8%) recipients were clinically stable. Of the 29 LTRs who deteriorated, MSSA pneumonia was confirmed in two patients. One received 14 days of vancomycin, and the sec ond patient was on cefazolin since transplantation, which was switched to cloxacillin after a result of 2week BAL due to pro gression of pneumonia. Both patients achieved full recovery. The causes of deterioration in the other 27 LTRs were attrib uted to nonMSSA pneumonia (14 out of 27), of which 6 were due to P. aeruginosa pneumonia, acute rejection (6 out of 27), or myocardial infarction (3 out of 27); while four LTRs were treated for both rejection and pneumonia. Eventually two of the patients who developed myocardial infarction died. During the following 10 weeks of the observation period, an additional six recipients had MSSAconfirmed infection. Five of them had MSSA pneumonia, while one patient had concomitant MSSA pneumonia and bacteremia. The latter patient received 4 weeks of cefazolin for persistent MSSA bacteremia, but infective endocarditis was ruled out by transesophageal echocardiogram. Eventually, he achieved eradication of the blood stream infection and was dis charged home. The other five patients received 2 weeks of different antimicrobial agents including cefazolin, piperacil lintazobactam, and amoxicillinclavulanic acid, and they all achieved full recovery. None of the recipients developed MSSArelated anastomotic infection, empyema, lung abscess, or infective endocarditis during the followup time. The details of the patient characteristics, treatment, and clinical outcomes in the recipients who developed MSSA infection are shown in Table 4.

Discussion
We conducted a retrospective evaluation of the rate of micro biological eradication of MSSA isolated in BAL fluid at the time of transplant with followup to 12 weeks after lung transplant. We also looked at the development of MSSA infection and clinical outcomes in these recipients up to 12 weeks after transplant.
This study demonstrated a high rate of MSSA eradication at the 2week posttransplant bronchoscopy in the LTRs irre spective of it being of donor or/and recipient origin. To the best of our knowledge, our study is the first of its kind evalu ating the microbiological outcomes associated with MSSA isolated in the respiratory cultures of LTRs perioperatively. Interestingly, more recipients had positive respiratory cul tures for MSSA at 6week (5 of 173) and 12week (7 of 174) bronchoscopy than at the 2week bronchoscopy assessment (3 of 189). This could be due to the fact that most patients had been treated with antibiotics within the 2week time frame prior to the 2week bronchoscopy, which may have contributed to the negative respiratory cultures. At the 2week and 6week assessment bronchoscopies, LTRs with positive respiratory cultures were more likely to have a simi lar strain of MSSA than positive cultures at 12 weeks, when acquisition of new MSSA strains contributed to a 57% posi tivity rate of the MSSA in respiratory cultures (4 of 7). However, the overall rate of eradication remained high up to the 12week evaluation (96.1%).
Previous studies have demonstrated that S. aureus is the second most common organism isolated in lung transplants perioperatively, with 74% of the isolates being MSSA. 12 However, whether LTRs with positive MSSA cultures at the time of transplant will subsequently progress to clinically significant infection is unclear. We demonstrated a low prev alence of MSSArelated infection in those patients. Over the 12week study period, only 8 (4.2%) recipients were diag nosed with MSSA pneumonia, and concurrent MSSA bacte remia was diagnosed in only one of these patients (0.5%). Shields et al. 9 reported that 18% of LTRs developed S. aureus infection in the first 90 days after transplant, and 62% of them were due to MSSA. Although they found that the pres ence of MRSA in recipient sterility cultures and nasal swabs at the time of transplant was indeed a risk factor for early S. aureus infection, no specific risk factors were identified for MSSA infection 9 . Our low rate of infectious complications due to MSSA compared with the previous cohort may reflect the results of aggressive antibiotic management approaches taken in our LTRs from the time of transplantation. We observed that more than half of LTRs were treated with a dual antibiotic therapy. This is likely owing to the presence of polymicrobial respiratory cultures with concomitant Pseudomonas and other gramnegative organisms (87 of 189, 46%). It should be noted that most of the recipients who received a dual antibiotic therapy had received cloxa cillin or cefazolin in combination with an antipseudomonal antibiotic (62 of 96, 64.6%). This practice is derived from studies on MSSA bacteremia, which indicate that treating MSSA bacteremia with piperacillintazobactam, vancomy cin, or ceftriaxone alone was associated with a poor out come 10,13,14 . However, there is limited evidence of the impact of using betalactam, other than antistaphylococcal beta lactam, in clinical outcomes of patients with MSSA pneumo nia. A metaanalysis by Whiddon et al. found that using ceftriaxone to treat MSSA pneumonia is associated with higher clinical failure than ceftaroline or ceftobiprole, which are highly active against S. aureus 16 . In LTRs, such data are lacking. However, in contrary, recent data showed that anti biotic appropriateness at the time of lung transplantation has no impact on 30day mortality, hospital length of stay, or intensive care unit length of stay 12 .
In our study, the mean duration of therapy was 12.5 days, which is considered a longer duration of antibiotic therapy. Previously, using a longcourse perioperative antibiotic regi men had no mortality benefit in LTRs with positive cultures compared to a shorter course 8 . On the other hand, the dura tion of antimicrobials for the treatment of grampositive infection in this population has been associated with devel oping drugresistant organisms 16 . Presently, there are no studies that delineate the optimal perioperative antibiotic regimen or the duration of therapy for patients with MSSA isolated in respiratory secretions at the time of lung trans plantation. More studies are needed to identify whether treat ment of polymicrobial flora isolated from the BAL fluid cultures that may include MSSA at the time of transplanta tion requires prolonged broadspectrum coverage, or whether a shorter duration would be adequate to diminish the threat of the emergence of resistance in keeping with the principles of antimicrobial stewardship.
Our study, however, has several limitations. The retro spective cohort design excluding patients who missed fol lowup or died prior to the 2week bronchoscopy hindered the assessment of the primary outcome and may have lim ited enrollment. Although none of the excluded recipients had MSSA infection as the cause of death, we still cannot comment on the shortterm mortality related to MSSA due to the limited number of patients. In addition, we excluded those individuals who had positive respiratory cultures for MRSA (isolated from the donor or recipient) as well as con comitant MSSA and MRSA due to concerns about biasing the primary endpoint results for MSSA as MRSA may be harder to eradicate. Moreover, as with all retrospective stud ies, missing data at times posed a problem. This prevented us from comparing antibiotic regimens between those included and those excluded. Third, due to the low number of patients who received piperacillintazobactam or merope nem monotherapy, we could not evaluate whether the use of a broadspectrum antibiotic alone versus an antistaphylo coccal betalactam may have exerted an impact on clinical or microbiological outcomes or on mortality associated with MSSA infection. Consistent with this, the number of recipients who developed clinical MSSA infection was low precluding any analysis of riskidentifying factors for MSSA infection.
In conclusion, MSSA is a commonly isolated organism in the respiratory secretion cultures of donors and recipients at the time of lung transplantation. The rate of eradication by 2 weeks was high and was associated with a low rate of infec tious complication within the first 12 weeks after transplant. Most of the recipients received a combination therapy with at least one agent active against MSSA. However, it was not clear whether this is an appropriate practice or not. More studies to evaluate the optimal regimen and its duration are needed to enhance antimicrobial stewardship in LTRs.